Many proteins, including recombinant proteins, may be isolated from milk for use as food additives and medicinal products, such as lactoferrin, glycomacropeptide (GMP), immunoglobulin, α-lactalbumin, β-lactoglobulin and lactoperoxidase. Casein in milk may be isolated for various industrial applications including paint and glue. Isolating a recombinant protein from the milk of transgenic livestock, however, is a challenging task because of the overabundance of casein, which constitutes 70% to 85% of total milk proteins.
Casein is relatively hydrophobic, making it poorly soluble in water. It is found in milk as a suspension of particles called casein micelles. The caseins in the micelles are held together by calcium ions and hydrophobic interactions. The casein micelle is difficult to be separated from whey in milk by using centrifugation or filtration method. Insoluble casein micelles create an obstacle for purification instruments, such as membrane filtration devices and chromatography columns, and cause termination of purification process. In addition, casein micelles precipitate only by using very high centrifugal force (50,000×g, or higher), which is not feasible for mass production in industry. In practice, purification of target protein from milk in large scale is usually hindered by the step of removing casein micelles.
The process of purification of milk proteins usually starts with casein removal. The conventional method precipitates caseins by acid precipitation at pH 4.6, in which the whey fraction, which contains most of proteins of interest, is recovered for further purification using chromatography techniques. The acid precipitation method is not applicable to acidic proteins such as human coagulation factor IX (hFIX), Hirudin and Erythropoietin because they have an isoelectric point (pI values) similar to caseins and thus are not separable from caseins under the acidic conditions. In addition, the low pH conditions used for casein removal usually leads to significantly poor yields and low biological activities.
In addition to acidification to pH 4.6, removal of casein micelles from milk can be facilitated by adding enzymes (such as chymosin) to destroy the structure of micelles, which may be accelerated by additional heat. The precipitated caseins and soluble proteins (whey proteins) are then separated by centrifugation or filtration. A lower pH value and additional heat can cause a permanent change in the protein structure and a loss in bioactivity. For instance, employing acid precipitation to transgenic goat's milk containing tissue-type plasminogen activator (tPA) causes a 50% loss of the tPA activity. Moreover, acidic recombinant proteins will precipitate under low pH conditions and will not separate from caseins. Morcol et al. reports that using acid precipitation to remove caseins in the process of purification of recombinant human coagulation factor IX (rhFIX) from transgenic ewe's milk resulted in a final total recovery of rhFIX only 2 to 2.5%. Polyethylene glycol (PEG) is also used to precipitate casein micelles for purification of protein in milk. The prerequisite for the method is that proteins such as recombinant proteins may not be precipitated by PEG and the protein content must be very high to overcome the losses during the process. U.S. Pat. No. 6,194,553 and EP 1115745 disclose a method for purification of α-1 protease inhibitor (α1-AT) from the milk of transgenic sheep, which involves two stages of PEG precipitation and five steps of chromatography column purification. PEG was first added to skim milk to produce casein precipitation for removal by centrifugation. The α1-AT remains in supernatant and the recovery yield at this step was 68%. A higher concentration of PEG was then added to precipitate α1-AT and other whey proteins, and the precipitate was purified using chromatography columns. This process has a low recovery yield.
Chelating agents for calcium, such as citric acid or EDTA, are used to form complex ions to dissolve casein micelles in milk, which are followed by an ultra-filtration process. U.S. Pat. No. 6,268,487 discloses a process of purifying antithrombin III (AT III) from the milk of transgenic goats using EDTA or citrate to increase the penetration rate of filtration. The method uses a large amount of solution to extract AT III, and the filtrate containing AT III and other proteins flow through an on-line affinity column to adsorb AT III protein. The recovery yield of this step was 75%, The product was subsequently applied to other chromatography columns for further purification to reach a medicine grade purity.
U.S. Pat. No. 6,355,271 and U.S. Pat. No. 6,183,803 disclose the use of artificial calcium phosphate-based particles (CAP) to remove casein micelles in milk, in which the structure of calcium micelles were solubilized by EDTA and salts (EDTA, calcium and phosphate ions, etc.) and micelles then removed by dialysis. The artificial calcium phosphate-based particles, manufactured by the BioSante company, were added into the casein-containing solution, and calcium phosphate particles associated with caseins formed a larger size of artificial casein micelles. The particles were precipitated by a lower centrifugal force (5000-10,000×g), and target protein and other whey proteins remain in the supernatant for further purification. The recovery yield was in the range of 90% to 95%, depending on the target proteins. The method involved extremely cumbersome steps and the method for preparing the calcium phosphate-base particles was very complicated.
The existing techniques for removing caseins from milk, has disadvantages such as restricted conditions, lower yields, damaged protein structures or loss in activities, inconvenience and difficulties in operation. Therefore, a previously unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies, especially in connection with the method for separating caseins from target protein in milk.